Bioactivation of xenobiotics to toxic intermediates through cytochrome P450 oxygenation mechanisms is a well recognized process. However, the production of electrophilic intermediates by several P450 enzymes (e.g. 1A2, 2B4, 2B6, 2C9, 2D6, 2E1, 2F1, 2F3, 2A13, and 3A4), through dehydrogenation pathways has only recently been investigated, and the mechanisms that govern selective dehydrogenation rather than oxygenation are not established. Several of the dehydrogenated intermediates are so reactive that they inactivate the P450 enzymes, generally through alkylation of active site nucleophilic residues. Recent convincing research on the P450 enzymes has documented the highly dynamic nature of these proteins that requires sophisticated computer-based simulations to model. Research concerning the catalytic behavior of these specific P450 enzymes and their propensity to dehydrogenate rather than oxygenate substrates is vitally needed. The hypothesis of this research is: the unique catalytic mechanism(s) of facilitated electron transport that determines dehydrogenation by certain P450 enzymes results in xenobiotic-mediated injury and altered drug metabolism in humans. The specific goals of this application are to determine the characteristics of the enzyme active-site and remote residue environments that direct dehydrogenation mechanisms of specific cytochrome P450 enzymes, and to define the substrate structural features that regulate selective dehydrogenation rather than oxygenation. These goals will be realized through the following aims: 1) to define the requisite chemical features of acceptable P450-mediated dehydrogenation substrates; 2) to characterize the chemical and biochemical mechanisms of dehydrogenation by evaluating the dehydrogenation of three prototype substrates, with the use of stable isotopes and identification of protein adducts; 3) to utilize integrated quantum mechanics-based models of substrates and intermediates with molecular mechanics and molecular dynamic simulations of P450 enzymes to predict critical dehydrogenation- specific residues and substrate reactivities; and 4) to validate specific P450 active site and remote residues by mutation of specific sites, followed by biochemical evaluations and x-ray structures of purified native and mutant enzymes. The long-term goals of this research are to elucidate the mechanisms of cytochrome P450- mediated dehydrogenation of xenobiotics in processes that generate toxic electrophilic intermediates, to assess the potential harm engendered by these toxic intermediates to human health, and to utilize mechanistic information to predict dehydrogenation, and concomitant toxicities and/or enzyme inactivation (altered drug metabolism), of new drugs and xenobiotics. PUBLIC HEALTH RELEVANCE: Medicines are chemicals that ideally have beneficial effects with few side effects, and that don't have drug/drug interactions, which is when they act together to cause the medicines to lose their efficacy. After a medicine is taken and has its beneficial action, it is usually metabolized, i.e. chemically altered, by cytochrome P450 enzymes in the liver to aid in its elimination. Even though the P450 enzymes generally convert the medicines to harmless metabolites that are excreted in the urine, frequently these enzymes change the structures of the medicines to highly reactive, toxic products, through a chemical mechanism called dehydrogenation. Dehydrogenation products are frequently toxic and cause drug/drug interactions by inactivating the P450 enzymes that metabolized the medicines. However, very little is known about the dehydrogenation process. Thus, the goal of this research is to precisely delineate the mechanisms of P450-mediated dehydrogenation, with medicines and toxic chemicals that are known to be metabolized by this process. Our long term objective is to predict which chemical motifs are likely to be dehydrogenation substrates, and should be avoided when new drugs are introduced. This knowledge will significantly improve the drug development process by the pharmaceutical industry in the future.